Wissenschaftliche Artikel der BAM
Filtern
Dokumenttyp
- Zeitschriftenartikel (52)
Sprache
- Englisch (52)
Schlagworte
- Cancer (6)
- Mycotoxins (5)
- Electrochemistry (4)
- Mass Spectrometry (4)
- Quality assurance (4)
- Reference material (4)
- Fluorescence (3)
- HRMS (3)
- Mass Spectroscopy (3)
- Metabolomics (3)
Organisationseinheit der BAM
- 1.7 Organische Spuren- und Lebensmittelanalytik (52) (entfernen)
Paper des Monats
- ja (1)
Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal tandards is challenging but currently carried out in the standard method EN 7425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N6-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (13CD3-I), to yield isotopically labeled ergotamine and its C8-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-13CD3 and ergotaminine-13CD3. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs.
We report an analytical methodology for the quantification of sulfur in biological molecules via a speciesunspecific postcolumn isotope dilution (online ID) approach using capillary electrophoresis (CE) coupled online with inductively coupled plasma−mass spectrometry (online ID CE/ICP−MS).
The method was optimized using a mixture of standard compounds including sulfate, methionine, cysteine, cystine, and albumin, yielding compound recoveries between 98 and 105%. The quantity of sulfur is further converted to the quantity of the compounds owing to the prior knowledge of the sulfur content in the molecules. The limit of detection and limit of quantification of sulfur in the compounds were 1.3−2.6 and 4.1−8.4 mg L−1, respectively, with a correlation coefficient of 0.99 within the concentration range of sulfur of 5−100 mg L−1. The capability of the method was extended to quantify albumin in its native matrix (i.e., in serum) using experimentally prepared serum spiked with a pure albumin standard for validation. The relative expanded uncertainty of the method for the quantification of albumin was 6.7% (k = 2). Finally, we tested the applicability of the method on real samples by the analysis of albumin in bovine and human sera. For automated data assessment, a software application (IsoCor) which was developed by us in a previous work was developed further for handling of online ID data. The method has several improvements compared to previously published setups: (i) reduced adsorption of proteins onto the capillary wall owing to a special capillary-coating procedure, (ii) baseline separation of the compounds in less than 30 min via CE, (iii) quantification of several sulfur species within one run by means of the online setup, (iv) SI traceability of the quantification results through online ID, and (v) facilitated data processing of the transient signals using the IsoCor application. Our method can be used as an accurate approach for quantification of proteins and other biological molecules via sulfur analysis in complex matrices for various fields, such as environmental, biological, and pharmaceutical studies as well as clinical diagnosis.
Sulfur is an essential element in living organisms, where it plays important roles in various biological processes, such as protein synthesis, enzyme activity, and antioxidant defense. However, the biological effects of different sulfur species can vary widely, and imbalances in sulfur speciation have been observed in a range of diseases, including cancer, Alzheimer’s disease, and diabetes.1−3 The accurate quantification of sulfur and its species in biological samples requires sensitive and selective analytical techniques. In recent years, separation techniques coupled online with inductively coupled plasma−mass spectrometry (ICP−MS) have emerged as powerful online analytical tools complementary to molecular spectrometric methods for speciation analysis of biological compounds.
External calibration4−9 and isotope dilution (ID)10−15 are common calibration approaches applied for online quantification of sulfur species in complex samples. The ID analysis is advantageous over.
Here, we report a comparative study of different sum parameter analysis methods for the extraction of per- and polyfluoroalkyl substances (PFAS) from manufactured consumer products, which can be measured by combustion ion chromatography (CIC). Therefore, a hydrolysis-based extraction method was further developed, which accounts for the addition of hydrolyzable covalently bound polyfluoroalkylated side-chain polymers (SFPs) to the extractable organic fluorine portion of the mass balance proposed as "hydrolyzable organically bound fluorine" (HOF). To test this hypothesis, the method was applied to 39 different consumer products containing fluoropolymers or monomeric PFAS taken from four different categories: outdoor textiles, paper packaging, carpeting, and permanent baking sheets. We also evaluated the method's efficiency by extracting four synthesized fluorotelomer polyacrylate reference compounds. The total fluorine (TF) and extractable organically bound fluorine (EOF) values were measured through CIC using established protocols. The TF values ranged from sub-ppb to %-levels, depending on the compound class. All samples showed results for hydrolyzed organofluorine (HOF) between 0.03 and 76.3 μg/g, while most EOF values were lower (<LOD to 58.1 μg/g). Moreover, the methods were contrasted with the "total hydrolyzable precursor" (THP) assay analyzed using gas-chromatography coupled mass spectrometry (GC-MS), which yielded concentrations of 6:2–10:2 fluorotelomer alcohols (<LOD-568 μg/g) for textile and paper samples. Furthermore, targeted analytical investigation of 25 compounds via liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) was conducted and accounted for significantly lower sum amounts (<LOD-0.98 μg PFAS/g). To estimate the fluorine mass balance of all methods, the fluorine levels of target analytical data were calculated and compared to the sum parameter values and evaluated. Finally, the efficiency of the extraction method was approximated by the recovery analysis of four selected SFP model compounds compared to their TF values, yielding 36.7–74.0% after extraction and 3.8–36.3% after processing the extract for the HOF sum parameter.
The statistical tool eCerto was developed for the evaluation of measurement data to assign property values and associated uncertainties of reference materials. The analysis is based on collaborative studies of expert laboratories and was implemented using the R software environment. Emphasis was put on comparability of eCerto with SoftCRM, a statistical tool based on the certification strategy of the former Community Bureau of Reference. Additionally, special attention was directed towards easy usability from data collection through processing, archiving, and reporting. While the effects of outlier removal can be flexibly explored, eCerto always retains the original data set and any manipulation such as outlier removal is (graphically and tabularly) documented adequately in the report. As a major reference materials producer, the Bundesanstalt für Materialforschung und -prüfung (BAM) developed and will maintain a tool to meet the needs of modern data processing, documentation requirements, and emerging fields of RM activity. The main features of eCerto are discussed using previously certified reference materials.
Ergot alkaloids are a group of toxic compounds, formed by fungi on infested grasses. In 2022, the European Commission set into effect maximum levels for the sum of the twelve major ergot alkaloids in multiple foods. To facilitate the laborious and costly individual quantification of the twelve major ergot alkaloids by HPLC–MS/MS or -FLD, we recently reported a sum parameter method (SPM) for ergot alkaloid quantification. Here, derivatization to lysergic acid hydrazide—a derivative of the mutual ergoline backbone in all ergot alkaloids—allowed simplified determination of all ergot alkaloids in flour via HPLC-FLD. For the measurement of more complex matrices like processed foods, we now developed a MS/MS-based SPM. Two internal standards (IS), isotopically labelled at different positions of the molecule, were synthesized and employed in the MS/MS-measurements. Method performance using either the 13CD3-labelled or the 15N2-labelled IS was evaluated on naturally contaminated rye and wheat flour samples as well as on processed food matrices. Employing the 13CD3-labelled IS leads to lower variances and better consistency with the reference data (obtained by the FLD-based SPM) in flour samples compared to the 15N2-labelled IS. The novel method significantly improves the measurement of ergot alkaloids in complex food matrices, due to their increased selectivity and thus lower interferences. Furthermore, the application of isotope labelled IS obviates the need for time-consuming steps like the determination of recovery rate based, matrix specific correction factors as described in the MS/MS-based European standard method for ergot alkaloid quantification (EN 17425).
Solid wastewater-based fertilizers were screened for per- and polyfluoroalkyl substances (PFAS) by the extractable organic fluorine (EOF) sum parameter method. The EOF values for ten sewage sludges from Germany and Switzerland range from 154 to 7209 mg kg−1. For thermal treated sewage sludge and struvite the EOF were lower with values up to 121 mg kg−1. Moreover, the application of PFAS targeted
and suspect screening analysis of selected sewage sludge samples showed that only a small part of the EOF sum parameter values can be explained by the usually screened legacy PFAS. The hitherto unknown part of EOF sum parameter contains also fluorinated pesticides, pharmaceutical and aromatic compounds. Because these partly fluorinated compounds can degrade to (ultra-)short PFAS in wastewater treatment plants they should be considered as significant sources of organic fluorine in the environment. The combined results of sum parameter analysis and suspect screening reveal the need to update current regulations, such as the German fertilizer ordinance, to focus not solely on a few selected PFAS such as perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) but
consider an additional sum parameter approach as a more holistic alternative. Moreover, diffusion gradient in thin-films (DGT) passive samplers were utilized as an alternative simplified extraction method for PFAS in solid wastewater-based fertilizers and subsequently quantified via combustion ion chromatography. However, the DGT method was less sensitive and only comparable to the EOF values
of the fertilizers in samples with >150 mg kg−1, because of different diffusion properties for various PFAS, but also kinetic exchange limitations.
For the first time, µ-X-ray fluorescence (µ-XRF) mapping combined with fluorine K-edge µ-X-ray absorption near-edge structure (µ-XANES) spectroscopy was applied to depict per- and polyfluoroalkyl substances (PFAS) contamination and inorganic fluoride in samples concentrations down to 100 µg/kg fluoride. To demonstrate the matrix tolerance of the method, several PFAS contaminated soil and sludge samples as well as selected consumer product samples (textiles, food contact paper and permanent baking sheet) were investigated. µ-XRF mapping allows for a unique element-specific visualisation at the sample surface and enables localisation of fluorine containing compounds to a depth of 1 µm. Manually selected fluorine rich spots were subsequently analysed via fluorine K-edge µ-XANES spectroscopy. To support spectral interpretation with respect to inorganic and organic chemical distribution and compound class determination, linear combination (LC) fitting was applied to all recorded µ-XANES spectra. Complementarily, solvent extracts of all samples were target-analysed via LC-MS/MS spectrometry. The detected PFAS sum values range from 20 to 1136 µg/kg dry weight (dw). All environmentally exposed samples revealed higher concentration of PFAS with a chain length >C8 (e.g. 580 µg/kg dw PFOS for Soil1), whereas the consumer product samples showed a more uniform distribution with regard to chain lengths from C4 to C8. Independent from quantified PFAS amounts via target analysis, µ-XRF mapping combined with µ-XANES spectroscopy was successfully applied to detect both point-specific concentration maxima and evenly distributed surface coatings of fluorinated organic contaminants in the corresponding samples.
Ergot alkaloids are a group of mycotoxins occurring in products derived from various grasses (e.g., rye) and have been regulated in the EU recently. The new maximum levels refer to the sum of the six most common ergot alkaloids in their two stereoisomeric forms in different food matrices. Typically, these twelve compounds are individually quantified via HPLC-MS/MS or -FLD and subsequently summed up to evaluate food safety in a time-consuming process. Since all these structures share the same ergoline backbone, we developed a novel sum parameter method (SPM) targeting all ergot alkaloids simultaneously via lysergic acid hydrazide. After extraction and clean-up, in analogy to the current European standard method EN 17425 (ESM) for ergot alkaloid quantitation, the samples were derivatized by an optimized hydrazinolysis protocol, which allowed quantitative conversion after 20 min at 100 °C. The new SPM was evaluated against another established HPLC-FLD-based method (LFGB) and the HPLC-MS/MS-based ESM using six naturally contaminated rye and wheat matrix reference materials. While the SPM provided comparable values to the ESM, LFGB showed deviating results. Determined recovery rates, limits of detection and quantification of all three employed methods confirm that the new SPM is a promising alternative to the classical approaches for ergot alkaloid screening in food.
Volatile organic compounds (VOCs) are of interest in many different fields. Among them are food and fragrance analysis, environmental and atmospheric research, industrial applications, security or medical and life science. In the past, the characterization of these compounds was mostly performed via sample collection and off-site analysis with gas chromatography coupled to mass spectrometry (GC-MS) as the gold standard. While powerful, this method also has several drawbacks such as being slow, expensive, and demanding on the user. For decades, intense research has been dedicated to find methods for fast VOC analysis on-site with time and spatial resolution. We present the working principles of the most important, utilized, and researched technologies for this purpose and highlight important publications from the last five years. In this overview, non-selective gas sensors, electronic noses, spectroscopic methods, miniaturized gas chromatography, ion mobility spectrometry and direct injection mass spectrometry are covered. The advantages and limitations of the different methods are compared. Finally, we give our outlook into the future progression of this field of research.
Accessions of one plant species may show significantly different levels of susceptibility to stresses. The Arabidopsis thaliana accessions Col-0 and C24 differ significantly in their resistance to the pathogen Pseudomonas syringae pv. tomato (Pst). To help unravel the underlying mechanisms contributing to this naturally occurring variance in resistance to Pst, we analyzed changes in transcripts and compounds from primary and secondary metabolism of Col-0 and C24 at different time points after infection with Pst. Our results show that the differences in the resistance of Col-0 and C24 mainly involve mechanisms of salicylic-acid-dependent systemic acquired resistance, while responses of jasmonic-acid-dependent mechanisms are shared between the two accessions. In addition, arginine metabolism and differential activity of the biosynthesis pathways of aliphatic glucosinolates and indole glucosinolates may also contribute to the resistance. Thus, this study highlights the difference in the defense response strategies utilized by different genotypes.
Despite numerous advantages offered by hyphenation of chromatography and electrokinetic separation methods with multicollector (MC) ICP-MS for isotope analysis, the main limitation of such systems is the decrease in precision and increase in uncertainty due to generation of short transient signals. To minimize this limitation, most authors compare several isotope ratio calculation methods and establish a multi-step data processing routine based on the precision and accuracy of the methods. However, to the best of our knowledge, there is no universal data processing tool available that incorporates all important steps of the treatment of the transient signals. Thus, we introduce a data processing application (App) IsoCor that facilitates automatic calculation of isotope ratios from transient signals and eases selection of the most suitable method. The IsoCor App performs baseline subtraction, peak detection, mass bias correction, isotope ratio calculation and delta calculation. The feasibility and reliability of the App was proven by reproducing the results from isotope analysis of three elements (neodymium, mercury and sulfur) measured on-line via hyphenated systems. The IsoCor App provides trackability of the results to ensure quality control of the analysis.
Phosphorus (P) recovery is obligatory for all sewage sludges with more than 20 g P/kg dry matter (DM) from 2029 in Germany. Nine wastewater treatment plants (WWTPs) were chosen to investigate variations of phosphorus contents and other parameters in sewage sludge over the year. Monthly sewage sludge samples from each WWTP were analyzed for phosphorus and other matrix elements (C, N, H, Ca, Fe, Al, etc.), for several trace elements (As, Cr, Mo, Ni, Pb, Sn) and loss of ignition. Among the nine WWTPs, there are four which have phosphorus contents both above and below the recovery limit of 20 g/kg DM along the year. Considering the average phosphorus content over the year, only one of them is below the limit. Compared to other matrix elements and parameters, phosphorus fuctuations are low with an average of 7% over all nine WWTPs. In total, only hydrogen and carbon are more constant in the sludge.
In several WWTPs with chemical phosphorus elimination, phosphorus fuctuations showed similar courses like iron and/or aluminum. WWTPs with chamber flter presses rather showed dilution efects of calcium dosage. As result of this study, monthly phosphorus measurement is highly recommended to determine whether a WWTP is below the
20 g/kg DM limit.
Current trends in materials and life sciences are flanked by the need to push detection limits to single molecules or single cells, enable the characterization of increasingly complex matrices or sophisticated nanostructures, speed up the time of analysis, reduce instrument complexity and costs, and improve the reliability of data. This requires suitable analytical tools such as spectroscopic, separation and imaging techniques, mass spectrometry, and hyphenated techniques as well as sensors and their adaptation to application-specific challenges in the environmental, food, consumer product, health sector, nanotechnology, and bioanalysis. Increasing concerns about health threatening known or emerging pollutants in drinking water, consumer products, and food and about the safety of nanomaterials led to a new awareness of the importance of analytical sciences. Another important driver in this direction is the increasing demand by legislation, particularly in view of the 17 sustainable development goals by the United Nations addressing clean energy, industry, and innovation, sustainable cities, clean water, and responsible consumption and production. In this respect, also the development of analytical methods that enable the characterization of material flows in production processes and support recycling concepts of precious raw materials becomes more and more relevant. In the future, this will provide the basis for greener production in the chemical industry utilizing recycled or sustainable starting materials.
This makes analytical chemistry an essential player in terms of the circular economy helping to increase the sustainability of production processes. In the life sciences sector, products based on proteins, such as therapeutic and diagnostic antibodies, increase in importance. These increasingly biotechnologically produced functional biomolecules pose a high level of complexity of matrix and structural features that can be met only by highly advanced methods for separation, characterization, and detection. In addition, metrological traceability and target definition are still significant challenges for the future, particularly in the life sciences.
However, innovative reference materials as required for the health and food sector and the characterization of advanced materials can only be developed when suitable analytical protocols are available. The so-called reproducibility crisis in sciences underlines the importance of improved measures of quality control for all kinds of measurements and material characterization. This calls for thorough method validation concepts, suitable reference materials, and regular interlaboratory comparisons of measurements as well as better training of scientists in analytical sciences.
The important contribution of analytical sciences to these developments is highlighted by a broad collection of research papers, trend articles, and critical reviews from these different application fields. Special emphasis is dedicated to often-overlooked quality assurance and reference materials.
The aim of this study was to identify specific truffle marker substances within the truffle aroma. The aroma profile of different truffle species was analyzed using static headspace sampling with gas chromatography mass spectrometry analysis (SHS/GC-MS). Possible marker substances were identified, taking the additional literature into account. The selected marker substances were tested in an experiment with 19 truffle dogs. The hypothesis “If trained truffle dogs recognize the substances as supposed truffles in the context of an experiment, they can be regarded as specific” was made. As it would be nearly impossible to investigate every other possible emitter of the same compounds to determine their specificity, this hypothesis was a reasonable approximation. We were interested in the question of what it is the dogs actually search for on a chemical level and whether we can link their ability to find truffles to one or more specific marker substances. The results of the dog experiment are not as unambiguous as could have been expected based on the SHS/GC-MS measurements. Presumably, the truffle aroma is mainly characterized and perceived by dogs by dimethyl sulfide and dimethyl disulfide. However, as dogs are living beings and not analytical instruments, it seems unavoidable that one must live with some degree of uncertainty regarding these results.
Rapid, cost-efficient, and eco-friendly methods are desired today for routine analysis of the Fusarium mycotoxin zearalenone (ZEN) in edible vegetable oils. Liquid chromatography with fluorescence detection (HPLC-FLD) is commonly used to reliably control the specified ZEN maximum levels, which requires efficient sample clean-up to avoid matrix interferences. Therefore, a highly selective extraction and clean-up method based on reversible covalent hydrazine chemistry (RCHC) using hydrazine-functionalized silica was developed. This efficient solid-phase extraction (SPE) involves reversible hydrazone formation of ZEN with the hydrazine moiety covalently bound to a solid phase. Optimal conditions were achieved with 1 mL SPE cartridges filled with 400 mg of hydrazine-functionalized silica. The developed RCHC-SPE method was validated in an interlaboratory comparison study (ILC) with twelve participants analyzing six edible vegetable oils with a focus on maize oils. The derived method parameters (ZEN recovery 83%, repeatability 7.0%, and reproducibility 18%) meet the performance criteria of Commission Regulation (EC) No 401/2006. The developed RCHC-SPE-based HPLC-FLD method allows the reliable quantification of ZEN in the range of 47–494 μg/kg for different types of edible vegetable oils, also for matrix-reach native oils. Due to the high efficiency, the significantly reduced matrix load helps to extend the lifetime of analytical equipment. Furthermore, the re-useability of the RCHC-SPE cartridges contributes to an eco-friendly approach and reduced analysis costs. To our knowledge, this is the first report on ZEN quantification in edible vegetable oils based on manual RCHC-SPE cartridges. Due to its high performance, the developed RCHC-SPE method is a promising alternative to the current European standard method EN 16924:2017 (HPLC-FLD part).
Analysis of electrochemical and liver microsomal transformation products of lasalocid by LC/HRMS
(2022)
Rationale: Lasalocid (LAS), an ionophore, is used in cattle and poultry farming as feed additive for its antibiotic and growth-promoting properties. Literature on transformation products (TP) resulting from LAS degradation is limited. So far, only hydroxylation is found to occur as the metabolic reaction during the LAS degradation. To investigate potential TPs of LAS, we used electrochemistry (EC) and liver microsome (LM) assays to synthesize TPs, which were identified using liquid chromatography high-resolution mass spectrometry (LC/HRMS).
Methods: Electrochemically produced TPs were analyzed online by direct coupling of the electrochemical cell to the electrospray ionization (ESI) source of a Sciex Triple-TOF high resolution mass spectrometer. Then, EC-treated LAS solution was collected and analyzed offline using LC/HRMS to confirm stable TPs and improve their annotation with a chemical structure due to informative MS/MS spectra. In a complementary approach, TPs formed by rat and human microsomal incubation were investigated using LC/HRMS. The resulting data were used to investigate LAS modification reactions and elucidate the chemical structure of obtained TPs.
Results: The online measurements identified a broad variety of TPs, resulting from modification reactions like (de-)hydrogenation, hydration, methylation, oxidation as well as adduct formation with methanol. We consistently observed different ion complexations of LAS and LAS-TPs (Na+; 2Na+ K+; NaNH4 +; KNH4 +). Two stable methylated EC-TPs were found, structurally annotated, and assigned to a likely modification reaction. Using LM incubation, seven TPs were formed, mostly by oxidation/hydroxylation. After the identification of LM-TPs as Na+-complexes, we identified LM-TPs as K+-complexes.
Conclusion: We identified and characterized TPs of LAS using EC- and LM-based methods. Moreover, we found different ion complexes of LAS-based TPs. This knowledge, especially the different ion complexes, may help elucidate the metabolic and environmental degradation pathways of LAS.
Lifestyle modifications could prevent almost one‑third to one‑half of all cancer cases. The awareness of cancer risk factors could motivate people to make such changes in their behaviors and lifestyles. This work aims to investigate the cancer awareness level in the Pakistani population. Telephone interviews of 657 individuals in Pakistan were carried out using the Cancer Awareness Measure (CAM) and Cancer Awareness Measure–MYthical Causes Scale (CAM‑MY). We observed that participants scored significantly better on the CAM scale than the CAM‑MY scale, and CAM scores were negatively associated with CAM‑MY scores. Years of formal education or a biology major at undergraduate or graduate level did not affect our population’s cancer awareness levels. Age displayed a weak but statistically significant negative association with CAM scores. Most participants failed to identify modifiable cancer risk factors, e.g., low physical activity. Efforts should be made to improve awareness of modifiable risk factors. We observed that brief training sessions could markedly improve people’s understanding of cancer risk factors and myths.
For more than 110 years, BAM has been producing reference materials for a wide range of application fields. With the development of new analytical methods and new applications as well as continuously emerging more stringent requirements of laboratory accreditation with regard to quality control and metrological traceability, the demand and requirements for reference materials are increasing. This trend article gives an overview of general developments in the field of reference materials as well as developments in selected fields of application in which BAM is active. This includes inorganic and metal analysis, gas analysis, food and consumer products, and geological samples. In addition to these more traditional fields of application, developments in the areas of optical spectroscopy, particulary fluorescence methods, and nanomaterials are considered.
Towards Unbiased Evaluation of Ionization Performance in LC-HRMS Metabolomics Method Development
(2022)
As metabolomics increasingly finds its way from basic science into applied and regulatory environments, analytical demands on nontargeted mass spectrometric detection methods continue to rise. In addition to improved chemical comprehensiveness, current developments aim at enhanced robustness and repeatability to allow long-term, inter-study, and meta-analyses. Comprehensive metabolomics relies on electrospray ionization (ESI) as the most versatile ionization technique, and recent liquid chromatography-high resolution mass spectrometry (LC-HRMS) instrumentation continues to overcome technical limitations that have hindered the adoption of ESI for applications in the past. Still, developing and standardizing nontargeted ESI methods and instrumental setups remains costly in terms of time and required chemicals, as large panels of metabolite standards are needed to reflect biochemical diversity. In this paper, we investigated in how far a nontargeted pilot experiment, consisting only of a few measurements of a test sample dilution series and comprehensive statistical analysis, can replace conventional targeted evaluation procedures. To examine this potential, two instrumental ESI ion source setups were compared, reflecting a common scenario in practical method development. Two types of feature evaluations were performed, (a) summary statistics solely involving feature intensity values, and (b) analyses additionally including chemical interpretation.
Results were compared in detail to a targeted evaluation of a large metabolite standard panel. We reflect on the advantages and shortcomings of both strategies in the context of current harmonization initiatives in the metabolomics field.
MALDI-TOF-MS-Based Identification of Monoclonal Murine Anti-SARS-CoV-2 Antibodies within One Hour
(2022)
During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 degrees C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.